A major goal in the forestry and paper industries is the control of lignin content in plants. Lignin is a complex polymer of cinnamyl alcohols that is responsible for wood's mechanical strength, coloration, and resistance to rot. Tree species synthesize large quantities of lignin, with lignin constituting between 20% to 30% of the dry weight of wood. In addition to providing rigidity, lignin aids water transport by rendering cell walls hydrophobic and water-impermeable. It follows that increasing the lignin concentration in trees can prove beneficial for certain applications, such as providing trees with improved disease resistance or increased strength for use in construction. Lignin is also useful as a fuel, and lignin together with increased cellulose content is desirable in wood or other biomass used as fuel, such as wood for charcoal production, corn stover, and trees such as willow and fast growing aspen hybrids used for biofuels.
Conversely, the high concentration of lignin in trees presents a significant problem for the paper industry, which must expend considerable resources to separate lignin from cellulose fiber. In the U.S. alone, about 20 million tons of lignin are removed from wood per year. Further, the content of lignin is largely responsible for the digestibility, or lack thereof, of forage crops, with small increases in plant lignin content resulting in relatively high decreases in digestibility. For example, crops with reduced lignin content provide more efficient forage for cattle, with the yield of milk and meat being higher relative to the amount of forage crop consumed. Lignin content increases during plant growth, so that farmers must choose between harvesting crops early to obtain a lower yield of more digestible crops or later to obtain a higher yield of less digestible material.
For these reasons, the control of lignin content or composition through genetic modification of plants is desirable. Considerable effort has been made to this end to identify and characterize the genes responsible for lignin biosynthesis and to determine sequences that regulate their expression. Polynucleotides encoding many of the enzymes involved in lignin biosynthesis have been cloned, including cinnamyl alcohol dehydrogenase (CAD), cinnamate 4-hydroxylase (C4H), coumarate 3-hydroxylase (C3H), phenolase (PNL), O-methyl transferase (OMT), cinnamoyl-CoA reductase (CCR), phenylalanine ammonia-lyase (PAL), 4-coumarate:CoA ligase (4CL) and peroxidase (POX) from pine. U.S. Pat. No. 6,204,434.
Manipulation of the expression of these genes has been used to modify lignin content. Such experiments include altering the number of copies of genes encoding CAD, coniferin β-glucosidase (CBG), and caffeic acid 3-O-methyltransferase (COMT). U.S. Pat. No. 5,451,514, WO 94/23044, and Dharmawardhana et al, Plant Mol. Biol. 40: 365-72 (1999). Furthermore, antisense expression of sequences encoding CAD in poplar, N. tabacum, and pine leads to the production of lignin having a modified composition. Grand et al., Planta (Berl.) 163: 232-37 (1985), Yahiaoui et al., Phytochemistry 49: 295-306 (1998), and Baucher et al., Plant Physiol. 112: 1479 (1996), respectively.
Another major goal of the forest products, paper, plant biomass and forage industries is to increase the size of the stem, to manipulate cellulose content in the stem, or to manipulate characteristics of the cell wall in order to facilitate the recovery of cellulose from the stem. For example, cellulose is recovered from the xylem fibers in pulp production, and the number of xylem fibers and vessel elements, thickness of the cell walls, diameter of the cell lumens, length of the fibers, cellulose microfibril angle and other characteristics of these xylem cells determine the quality and quantity of cellulose recovered. Manipulation of genes involved in cellulose biosynthesis has been useful to increase the total biomass of plants and the yield of cellulose from the plants, while antisense expression of such genes has demonstrated effects on cell wall development. Shani Z., Shpigel, E., Roiz, L., Goren, R., Vinocur, B., Tzfira, T., Altman, A., and Shoseyov O. Cellulose binding domain increases cellulose synthase activity in Acetobacter xylinum, and biomass of transgenic plants. In: A. Altman, M. Ziv, S. Tzhar, eds., Plant Biotechnology and In Vitro Biology in the 21st Century, pp. 213-218 Kluwer Academic Publishers. (1999). Modification of polysaccharides and plant cell wall by endo-1,4-β-glucanase and cellulose-binding domains has been described. Levy, I., Shani, Z. and Shoseyov O. Biomol Eng. 19: 17-30 (2002). Accordingly, the polynucleotides of the instant invention can be used to express nucleotide sequences in vascular tissue to modify cellulose biosysnthesis thereby affecting plant growth and biomass.
Genetic regulation of biochemical pathways preferably is conducted in narrowly restricted tissue types to avoid global, detrimental effects to the modified plants. For example, when the content or composition of lignin is affected by expression of a particular gene product, it may be desirable to limit the expression of the gene product to certain segments of the plant or to certain developmental stages, to avoid decreasing the plant's disease resistance. A heterologous gene may be expressed in a selected tissue by operably linking it to a tissue-preferred promoter. Suitable tissue-preferred promoters include the bean grp1.8 promoter, which is specifically active in protoxylem tracheary elements of vascular tissue. Keller et al., EMBO J. 8: 1309 (1989). These promoters also include the eucalyptus CAD promoter, which is preferentially expressed in lignifying zones. Feuillet et al., Plant Mol. Biol. 27: 651 (1995). Such tissue-preferred promoters have been used to regulate gene expression of antisense molecules in specific tissues. Van der Meer et al., Plant Cell 4: 253 (1992), Salehuzzaman et al., Plant Mol. Biol. 23: 947 (1993), and Matsuda et al., Plant Cell Physiol. 37: 215 (1996).
Because tissue-preferred promoters may be less active in a heterologous environment, they do not always express genes to the same levels achieved with constitutive promoters. Yahiaoui et al., Phytochemistry 49: 295-306 (1998). Further, the developmental window during which these promoters are active, or the spatial distribution of their activity, may limit their usefulness. Thus, there is a continuing need in the art to define additional tissue-preferred promoters, especially vascular-preferred promoters, that have desirable spatial and temporal patterns of expression. Reviewed by Grima-Pettenati et al., Plant Science 145: 51-65 (1999).